Methods of using and constructing nanosensor platforms

ABSTRACT

The present invention relates to the use of nanowires, nanotubes and nanosensor platforms. In one embodiment, the present invention provides a method of constructing a nanosensor platform. In another embodiment, the present invention provides a method of analyzing multiple biomarker signals on a nanosensor platform for the detection of a disease.

FIELD OF THE INVENTION

The invention relates to the field of biotechnology; specifically, to nanosensor platforms and detection of a disease and condition.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

A diversity of sensor architectures have been designed and fabricated during the last decade that utilize different nanomaterials as a sensing element (cantilevers, quantum dots, nanotubes, nanowires, nanobelts, nanogaps, and nanoscale films). Some of these sensing devices, such as those based on cantilevers and quantum dots are highly specific, ultra sensitive, and have short response times. However, many of these devices require integration with optical components in order to translate surface binding phenomena into a readable signal. The need for detection optics is expected to significantly increase the cost of operation for such a device. Thus, there is a need in the art for alternative sensing devices.

As known in the art, the development of a disease, bacterial or viral contamination, allergic reaction, etc. in a subject can also result in the presence of related biomarkers. In turn, these diseases and/or conditions can be monitored by tracking the presence of biomarkers. For example, there are now several known biomarkers of cancer. During the last few years, the molecular basis for cancers has been increasingly elucidated, and common as well as distinct genetic alterations involved in the multi-step mechanism of carcinogenesis are beginning to be understood, thereby offering additional markers for cancer.

However, for most molecular detection techniques that have been developed and validated for their diagnostic and prognostic utility, efficiency is a result of a trade-off between sensitivity, specificity, ease of operation, cost, speed and availability to routine clinical laboratories. These include time-tested manual techniques, as well as a number of automated techniques such as microfabricated arrays and surface plasmon resonance systems. Thus, there is a need in the art for additional molecular detection techniques that are efficient, as well as effective.

Similarly, there is a need in the art for multiple marker assays. Serum is a source of protein and nucleic acid biomarkers, and, by its very nature, can reflect organ-confined events. Use of single biomarkers provides only a limited sensitivity and specificity for detection. Prostate specific antigen (PSA) for prostate cancer screening, for example, is one of the few examples of a single cancer marker that has been used in cancer screening, and it has been shown to be problematic due to its lack of specificity. In contrast, use of a multi-marker approach has been shown to add specificity and sensitivity to screening. Many diseases and conditions, such as cancer, are characterized by a number of molecular alterations and make multiple marker assays critical for successful, early detection of the disease and/or condition. Because of recent advances in quantitative methods and biomarker development, such as matrix assisted laser desorption ionization-time of flight-mass spectroscopy, surface enhanced laser desorption ionization, capillary electrophoresis, 2D-GE, surface plasmon resonance, etc., there have been many discoveries of multiple markers for a condition. It has now become possible to generate a molecular pattern map so as to achieve the goal of cancer screening, for example, using serum tests. The availability of an inexpensive assay platform for detecting markers in a sensitive, and specific high-throughput manner would therefore be highly desirable.

For example, it has been shown that simultaneous quantitation of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II) can discriminate between disease-free and epithelial ovarian cancer patients, including patients diagnosed with stage I and II disease, with high (95%) efficiency. While a single protein may not completely distinguish the cancer group from the healthy controls, the combination of the four analytes exhibited high sensitivity, positive predictive value, specificity, and negative predictive value. This example demonstrates the utility and significance of multiplex biomarker detection and quantitation. There is the potential for solving problems associated with screening for low prevalence cancers, for example, like ovarian cancer, where single markers as individual determinants have limited or no value for screening. There is also the need to develop single-platform technologies for multiplex marker detection, which can obviate the problems of high costs and multiple laborious interventional steps associated with current techniques such as ELISA. Additionally, there is a need to develop a screen for multiple different conditions and diseases, such as testing for single low-prevalence cancers, where even if specific it will be cost-prohibitive. A single, low-cost test, such as that involving a chip for detection of markers for multiple cancers, will thus prove advantageous.

SUMMARY OF THE INVENTION

Various embodiments provide a nanosensor comprising a nanomaterial configured for electrical signaling, and one or more capture agents distributed on a surface of the nanomaterial, where the nanosensor is configured such that binding of a target molecule to one of the one or more capture agents causes a change in electrical signaling. In another embodiment, the change in electrical signaling is a change in conductance, current, transconductance, capacitance, threshold voltage, or combinations thereof. In another embodiment, the capture agent comprises a polynucleotide and/or polypeptide. In another embodiment, the capture agent comprises an aptamer, a receptor, a ligand, or a combination thereof. In another embodiment, the nanomaterial comprises a carbon nanotube. In another embodiment, the nanomaterial is fabricated by patterned growth of carbon nanotubes. The nanomaterial may also comprise an In₂O₃ nanowire. In another embodiment, the change in transconductance is calibrated by liquid gate measurement. In another embodiment, the target molecule comprises an analyte. In another embodiment, the target molecule comprises a biomolecule. In another embodiment, the presence or absence of the biomolecule is indicative of a molecular signature associated with a disease.

Other embodiments include a complementary detection system, comprising an orthogonal functionalization of a nanomaterial with a substrate. In another embodiment, the nanomaterial comprises a carbon nanotube and/or an In₂O₃ nanowire. In another embodiment, the substrate comprises Si/SiO₂.

Other embodiments include a method of preparing a biosensor to detect the presence of a molecular signature associated with a disease, comprising providing a biosensor comprising one or more pairs of interdigitated source and drain electrodes, and fabricating a plurality of nanowires on the one or more pairs of interdigitated source and drain electrodes. In other embodiments, the nanowire comprises In₂O₃. In other embodiments, the IN₂O₃ was grown on a substrate. In other embodiments, the one or more interdigitated source and drain electrodes each have a channel length of between 1 micron and 100 microns and a channel width of between 100 microns and 1000 microns. In other embodiments, the interdigitated source and drain electrodes each have a channel length of about 2.5 microns and a channel width of about 500, about 780, and/or about 2600 microns.

Other embodiments include a method of preparing a biosensor array, comprising placing a quantity of poly-silicon and/or a quantity of amorphous-silicon on an insulating substrate, and incorporating the quantity of poly-silicon and/or the quantity of amorphous-silicon as a component of the biosensor array.

Other embodiments may include a biosensor array, comprising a thin-film semiconductor patterned into one or more nanowires.

Other embodiments include a nanosensor platform, comprising a field effect transistor configured with a plurality of interdigitated electrodes and nanowire, and a poly/amorphous silicon-on-insulator where the poly/amorphous silicon-on-insulator is a component of the field effect transistor. In another embodiment, the nanowire comprises In₂O₃.

Various embodiments include a method of fabricating a nanotube biosensor, comprising preparing a catalyst, growing aligned nanotubes by utilizing prepared catalyst; and defining metal electrodes separated by the aligned nanotubes. In another embodiment growing aligned nanotubes comprises a chemical vapor deposition growth of the nanotube with methane, ethylene, hydrogen and/or CO as feedstock. In another embodiment, growing aligned nanotubes comprises using sapphire and/or quartz as a substrate.

Other embodiments include a method of attaching elastomer polydimethylsiloxane to a silicon/silica surface, comprising treating the silicon/silica surface with a linker molecule, and attaching the elastomer polymethylsiloxane to the silicon/silica surface. In another embodiment, the linker molecule comprises silicic acid and/or alkyl silane. In another embodiment, the linker molecule comprises a silicon compound.

Various embodiments include calibrating the response of a nanosensor platform, comprising extracting one or more electronic properties of the nanosensor platform, and calibrating the response from the one or more electronic properties extracted from the nanosensor platform. In another embodiment, one of the one or more electronic properties comprises transconductance. In another embodiment, transconductance is defined by dividing by dIds/dVg. In another embodiment, the transconductance is defined by dividing by dlogIds/dVg.

Various embodiments include an apparatus for detecting and/or monitoring a disease, comprising a nanomaterial, and a plurality of capture molecules bound to the nanomaterial, where the plurality of capture molecules are configured for recognizing one or more biomolecules associated with a molecular signature of the disease. In another embodiment, the capture molecule comprises a polypeptide. In another embodiment, the capture molecule comprises a polynucleotide. In another embodiment, the capture molecule comprises an aptamer. In another embodiment, the capture molecule comprises a polynucleotide complex.

Other embodiments include a method of determining the presence of a disease in an individual from whom a sample is obtained, comprising removing background noise from the sample, providing a nanosensor device configured to detect the presence or absence of a multimarker signature of the disease, and contacting the nanosensor device with the sample to determine the presence or absence of the multimarker signature of the disease, where the presence of the multimarker signature is indicative of the disease. Other embodiments include removing the background noise by functionalization of the nanosensor device with one or more molecules that prevent binding of a nontarget entity. Other embodiments include removing the background noise by amplifying binding signals of the nanosensor device. Other embodiments include amplifying binding signals of the nanosensor device using a sandwich assay. Other embodiments include removing the background noise by preprocessing the sample to remove a major interfering component.

Other embodiments include a method of treating a disease, comprising providing a nanosensor device configured to detect the presence or absence of a molecular signature of the disease, contacting the nanosensor device with the sample to determine the presence or absence of the molecular signature of the disease, and treating the disease.

Various embodiments also include a method of improving the sensitivity of a nanosensor, comprising providing a nanosensor, and performing biosensing measurements by liquid gate voltage and/or back gate voltage to improve the sensitivity of the nanosensor.

Other embodiments include a method of preparing a biosensor array, comprising placing a quantity of semiconductor film on a substrate, and incorporating the quantity of semiconductor film as a component of the biosensor array. In other embodiments, the semiconductor film comprises single-crystal silicon. In another embodiment, the semiconductor film comprises poly-crystal and/or amorphous silicon.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with an embodiment described herein: (a) Optical micrograph of devices on complete 3″ wafer. (b) optical micrograph of channel area with interdigitated source and drain electrodes. (c) SEM image of an In₂O₃ NW between the source and drain electrodes. (d) family of source-drain current (I_(ds)) versus source-drain voltage (V_(ds)) plot under different gate voltage (V_(g)). The step of V_(g) is 1 V. (e) I_(ds) versus V_(g) plots in linear (left) and log (right) scale.

FIG. 2 depicts, in accordance with an embodiment described herein, SEM image of In₂O₃ nanowires dispersed on a Si/SiO₂ substrate with (a) high density and (b) low density. Statistical analysis of device yield with (c) high density nanowires and (d) low density nanowires.

FIG. 3 depicts, in accordance with an embodiment described herein, a histogram of threshold voltage of In₂O₃ nanowire devices on a Si substrate capped with 50 nm SiO₂.

FIG. 4 depicts, in accordance with an embodiment described herein, a process of making top-down fabrication of silicon nanowire using poly/amorphous silicon-on-insulator. (a) depicts poly-silicon or amorphous-silicon 100 deposited on appropriate substrates, such as dielectric 101 and silicon 102; (b) depicts active layer mesa defined using photolithography and reactive ion etching (RIE); (c) depicts ion implantation done to create degenerate lead-in, and doped region 103; (d) depicts annealing done to activate the doped region 103; (e) depicts e-beam writing and RIE is used to define nanowires with desired width; (f) depicts metal contacts 104 created using photolithography and lift-off technique.

FIG. 5 depicts, in accordance with an embodiment described herein, schematic diagram of fabrication of biosensor arrays using aligned carbon nanotubes. (a) depicts catalyst 105 preparation; (b) depicts aligned carbon nanotube 106 growth (c) depicts metal electrode 107 definition.

FIG. 6 depicts, in accordance with an embodiment described herein, images of biosensor array using aligned carbon nanotubes. (a) Schematic diagram of biosensor array with interconnections to bonding pads. (b) Optical micrograph of a biosensor chip. (c) SEM image of the region highlighted in (b). (d) SEM image of the active channel region of the devices. (e) SEM image of aligned nanotubes between source and drain electrodes.

FIG. 7 depicts, in accordance with an embodiment described herein, (a) histogram of aligned nanotube device resistance, and (b) IgG detection using an anti-IgG antibody decorated aligned nanotube biosensor, where the inset shows the schematic diagram of the device structure.

FIG. 8 depicts, in accordance with an embodiment described herein, typical density of CNTs that yields high on/off ratio devices.

FIG. 9 depicts, in accordance with an embodiment described herein, typical electrical characteristics where source-drain current (Ids) versus source-drain voltage (Vds) under different gate voltage (Vg) are plotted. I-Vgf and I-Vdsf. The clearly separated curves indicate the good sensitivity of the device to the gate voltage, which led to improved sensitivity of the device. (a) depicts Vds(V); (b) depicts Vg(V).

FIG. 10 depicts, in accordance with an embodiment described herein; the sensing of SA with a device using semiconductive nanotube network. The device showed ˜1% conductance drop upon exposure to a solution of SA at 100 pM, and further addition of SA at higher concentrations gave large conductance drops as shown (a). On the other hand, a device using mixed nanotube network showed only negligible response (<<1%) when exposed to 2 nM SA as shown (b), indicating that the lowest detection limit of mixed nanotube network devices is at least lower by a factor of 20 compared to semiconductive nanotube network devices. Furthermore, the comparison of magnitude of responses to SA at different concentrations (c) revealed the enhanced sensitivity of semiconductive nanotube network devices over mixed nanotube network devices, confirming the advantage of the use of semiconductive nanotube network as biosensors.

FIG. 11 depicts, in accordance with an embodiment described herein, the general structure of a bifunctional molecule. (a) depicts an example of a bifunctional molecule, as described herein. Similarly (b) depicts an example of a bifunctional molecule as described herein. For example, the molecule may be SiCl_(4;) or for example, the molecule may be Si(OR)₄ where R is alkyl or H.

FIG. 12 depicts, in accordance with an embodiment described herein, (a) a schematic diagram of the measurement setup, where to extract parameters used to calibrate sensor responses, liquid gate measurement was employed; with (b) depicting a typical Ids-Vg curve.

FIG. 13 depicts, in accordance with an embodiment described herein, typical Ids-Vg curves before/after the exposure of the device to a solution of Streptavidin, with an inset of a SEM image, showing. Streptavidin molecules tagged with 10 nm Au nanoparticles captured by an In₂O₃ nanowire functionalized with biotin.

FIG. 14 depicts, in accordance with, an, embodiment described herein, the successful normalization of the absolute response and relative response by dividing them by dIds/dVg and dlogIds/dVg, respectively. (a) shows absolute responses plotted against device identification number together with an average of the response before the calibration. Compared to (a), the normalized device responses (absolute response/dIds/dVg) showed smaller deviation from the average as shown in (b), which is after performing the calibration. The smaller deviation was verified by taking coefficient of variation (CV), which is one standard deviation divided by the mean. (c) shows CV for the absolute and relative responses before/after the calibration for Streptavidin and Avidin, respectively. The decreased CV after calibration for every response is depicted.

FIG. 15 depicts, in accordance with an embodiment described herein, (a) the sensing of Streptavidin (SA) when Vg=0.4 V. When the device was exposed to SA of 10, 100, and 1,000 nM, the device showed increased normalized conductance by 1, 2, and 10%, respectively. On the other hand, (b) with Vg=0.1 V, the device showed increased normalized conductance by 4, 24, and 47%, respectively, indicating the increased response.

FIG. 16 (prior art) depicts the structure of some intercalating molecules. (a) depicts YOYO 1: (b) depicts TOTO 1; (c) depicts POPO 1.

FIG. 17 depicts, in accordance with an embodiment described herein, a schema of peptide nucleic acids binding modes for targeting double-stranded DNA.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

As used herein, “FET” means field effect transistor.

As used herein. “NT” means nanotube.

As used herein, “NW” means nanowire.

As used herein, “SOI” means Silicon-On-Insulator.

As used herein, “RIE” means reactive ion etching.

As used herein, “CVD” means chemical vapor deposition.

As used herein. “DI” means de-ionized.

As used herein, “CNT” means carbon nanotubes.

As used herein, “PMMA” means poly(methyl methacrylate).

As used herein, “PDMS” means elastomer polydimethylsiloxane.

As used herein, “CV” means coefficient of variation.

As used herein, “SA” means Streptavidin.

As used herein, “PNA” means peptide nucleic acid.

As used herein, “aptamers” are molecules that may bind to a target molecule with specificity.

As used herein, “top-down” fabrication of nanowire technologies start with bulk materials and reduce the material dimensions using various techniques to cut, pattern, etch, and shape these materials into the desired geometry and order. In contrast, the “bottom-up” approach involves preparing the nanowire from molecular precursors, rather than starting with the bulk semiconductor.

As used herein, “background noise” refers to false or disruptive signals received when performing an assay to determine the presence or absence of analyte or a molecule of interest. For example, background noise may result when an analyte possesses a low net charge. Or, for example, when the analyte is present at trace levels in a complex mixture of biomolecules, such as blood, serum or urines.

As used herein, “intercalating” refers to the ability to insert into an existing structure, such as a polynucleotide sequence.

“Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to achieve beneficial results even if the treatment is ultimately unsuccessful.

I. Nanosensor Platforms

As disclosed herein, the inventors created nanobiosensors that are based on nanoscale FETs. The inventors prepared the PEI with a desired nanomaterial between source and drain electrodes, and then coated the nanomaterial with a molecular coating designed to bind a specific biomolecule or analyte. The binding of the target biomolecule and/or analyte to the nanobiosensor leads to a significant change in the environment surrounding the nanowire and/or nanotube, and a change in the transconductance of the device is detected. The high sensitivity for the device stems from the high surface to bulk ratio for nanowires and nanotubes, such that a small amount of biomolecule or analyte binding leads to a significant shift in the electronic properties of the nanoscale semiconductor.

As further disclosed herein, the inventors utilized complementary detection based on two material systems: carbon nanotubes and In₂0₃ nanowires. The inventors based their choice of nanomaterials on the need to achieve differential functionalization of the active sensing nanomaterials and the supporting substrates. As Si/SiO₂ substrates are the substrates of choice for most biosensing applications, using silicon nanowires would preclude selective functionalization of Si nanowires and Si/SiO₂ substrates with different functional groups, and an unwanted consequence is that antibodies and antigens, very often expensive and of minute quantity, would bind to both the nanowire and substrates. In contrast, orthogonal functionalization of carbon nanotubes, In₂O₃ nanowires and Si/SiO₂ substrates can be easily achieved. As a result, one can easily obtain selective functionalization of carbon nanotubes or In₂O₃ nanowires with the desired probe molecules, while the substrate can be passivated with other molecules to suppress nonspecific binding. One aspect of the invention includes passivating the substrate and surrounding channel at the upper stream of the microfluidic channel with antibodies and aptamers that can bind to the high-concentration proteins in serum samples that are not relevant to cancer detection, therefore effectively providing filtration of the serum sample before arriving at the nanosensors.

In one embodiment, the present invention provides a nanobiosensor where a nanomaterial is placed between a source and drain electrode. In another embodiment, the nanobiosensor is based on a nanoscale FET. In another embodiment, the nanomaterial is a nanowire and/or nanotube. In another embodiment, the nanomaterial is a carbon nanotube and/or In₂0₃. In another embodiment, the nanomaterial is coated with a molecular coating. In another embodiment, the molecular coating is designed to bind a specific biomolecule and/or analyte. In another embodiment, the binding of the target biomolecule and/or analyte causes a change in the environment surrounding the nanomaterial. In another embodiment, the binding of the target biomolecule and/or analyte causes a change in transconductance. In another embodiment, the binding of the target biomolecule and/or analyte causes a significant shift in the electronic properties of the nanoscale semiconductor. In another embodiment, the nanobiosensor utilizes a complementary detection system. In another embodiment, the complementary detection system undergoes an orthogonal functionalization of carbon nanotubes, In₂O₃ nanowires and/or Si/SiO₂ substrates. In another embodiment, the nanosensor uses interdigitated electrodes. In another embodiment, there is a filtration of a sample before arriving at the nanobiosensor.

As is readily apparent to one of skill in the art, any number of molecular coatings that are designed to bind a specific biomolecule or analyte may be used in conjunction with the various embodiments described herein, such as a polynucleotide, polypeptide, antibody, PNA, aptamer, receptor, ligand, or any number of combinations thereof. For example, aptamers offer an advantage of being small in size and thus changes in charge distribution due to binding of target molecules can occur closer to the surface of the nanobiosensor and thus increase the signal intensity. In comparison to antibodies, aptamers may tolerate harsher environmental changes which could result in longer shelf life and tolerate a wide variety of functionalization procedures.

Nanosensor Platforms—Nanowire Sensor Fabrication

As described herein, the inventors used interdigitated electrodes to achieve an easy and reliable fabrication of nanowire field effect transistor biosensors with improved uniformity and yield, as compared to devices fabricated with non-interdigitated electrodes. As is known to one of skill in the art, in order to perform quantitative analysis, an analysis necessary to monitor the concentration of biomarkers associated with the progress of many diseases, it is essential that there is a high yield of acceptable performing devices and uniform device performance (little device to device variation). Here, the inventors employed interdigitated electrodes to increase the effective channel width for the fabrication of nanobiosensor using a bottom up approach. Examples are depicted in FIGS. 1, 2 and 3. These interdigitated electrodes resulted in increasing the probability/number of the randomly dispersed nanowires to bridge between source and drain, while keeping the same footprint. As a consequence, devices fabricated with interdigitated electrodes have a more uniform performance with higher yield compared to devices with non-interdigitated electrodes, and the yield and uniformity of the devices are comparable to those achieved with assembling assisted techniques. This method has numerous advantages over the existing prior art, such as simplicity, scalability, reproducibility, high throughput, and room temperature processing.

In one embodiment, the present invention provides a nanowire biosensor with interdigitated electrodes. In another embodiment, the interdigitated electrodes increase the probability and/or number of nanowire to bridge between source and drain.

In another embodiment, the present invention provides a method of fabricating a nanowire biosensor by the following steps, or any combination thereof: (1) In₂O₃ nanowire is suspended in isopropanol by sonication; (2) the solution is dispersed onto a complete 3″ Si/SiO₂ substrate, followed by definition of Ti/Au source and drain electrodes by photolithography. In another embodiment, the In₂O₃ nanowire is previously grown on a Si/SiO, substrate via a laser ablation process. In another embodiment, the interdigitated electrodes have a channel length of 1 micron to 20 microns, with a preferred length of about 2.5 microns. In another embodiment, the interdigitated electrodes have an effective channel width of 10 microns to 5000 microns. In another embodiment, the interdigitated electrodes have an effective channel width of about 500 microns, 780 microns and/or 2600 microns.

As further disclosed herein, the inventors used a top-down fabrication of silicon nanowire using poly/amorphous silicon-on-insulator. An example is depicted in FIG. 4. By replacing single crystal silicon in a SOI wafer with poly-silicon and amorphous silicon, the inventors are able to reduce the cost significantly, while keeping the advantage of top-down, foundry compatible fabrication that results in high yield and small device-to-device variation. Furthermore, amorphous-silicon can be deposited on unconventional rigid/flexible substrates such as glass and PET, which further reduces the cost of production.

In one embodiment, the present invention provides a nanowire biosensor with a poly-silicon and/or amorphous silicon in a silicon-on-insulator wafer. In another embodiment, the amorphous silicon is deposited on an unconventional substrate. In another embodiment, the unconventional substrate is glass and/or PET.

In another embodiment, the present invention provides a top-down fabrication of silicon nanowire using three kinds of samples: single-crystal silicon-on-insulator, polysilicon film deposited on a substrate, and amorphous silicon film deposited on a substrate.

In another embodiment, the present invention provides a method of fabricating a nanowire biosensor by the following steps, or any combination thereof: (1) Poly-silicon and/or amorphous-silicon is deposited on an appropriate substrate, or single-crystal silicon-on-insulator wafer is used as the starting substrate; (2) an active layer mesa is defined using photolithography and reactive ion etching; (3) ion implantation is done to create degenerate lead-in; (4) annealing is performed to activate the dopants; (5) c-beam writing and reactive ion etching is used to define nanowires with the desired width; (6) metal contacts are created using photolithography and lift-off technique.

In another embodiment, the present invention provides a method of fabricating a nanowire biosensor by patterning a semiconductor film deposited on a substrate into nanowires via patterning and etching. In another embodiment, the semiconductor film may include single-crystal silicon, polysilicon, amorphous silicon, or materials such as GaAs, InP, and/or GaN.

Nanosensor Platforms—Nanotube Sensor Fabrication

As disclosed herein, the inventors developed a novel and straightforward approach for manufacturable and scalable biosensor arrays based on patterned growth of aligned carbon nanotubes at desired locations. An example is depicted in FIGS. 5, 6 and 7. This approach has several advantages, such as the capacity for mass production due to the use of conventional fabrication processes without e-beam writing; uniform and reproducible device performance due to the use of multiple nanotubes; and deterministic construction of biosensor arrays at specific locations and at any array size. Use of ordered nanotube arrays offers many important advantages, since the orientation control eases and increases the reproducibility of the sensor array fabrication. Furthermore, the architecture is fault tolerant: the destruction of one channel leaves other channels open, providing a conduction pathway between source and drain. This approach to nanosensor array fabrication may be used as the basis for a multiplexed nanobiosensor array fabrication.

In one embodiment, the present invention provides a biosensor array that utilizes highly aligned nanotubes as a semiconductor. In another embodiment, the nanotubes are single walled carbon. In another embodiment, the nanotubes are used for the detection of biomolecules. In another embodiment, the nanotube biosensor may be used to detect IgG antigen. In another embodiment, the nanotube biosensor may be functionalized with anti-IgG antibody by soaking in a solution of anti-IgG antibody in PBS buffer for about 12 hours at 4° C.

In another embodiment, the present invention provides a method of fabricating a nanotube biosensor by the following steps, or any combination thereof: (1) catalyst preparation; (2) aligned carbon nanotube growth; and (3) metal electrode definition. In another embodiment, the catalyst preparation includes one or more of the following: Quartz substrates are photolithographically patterned to make openings for catalysts; a solution of ferritin (Sigma) in de-ionized (D.I.) water is dropped onto the substrates, and kept for 10 min; the substrates are rinsed with D.I. water, and the photoresist layer is lifted off in acetone; the substrate with ferritin particles is calcinated at 700° C. for 10 min to form iron oxide nanoparticles that act as catalysts. In another embodiment, the aligned carbon nanotube growth includes a chemical vapor deposition (CVD) growth of CNTs with 2,500 sccm of Methane, 10 sccm of Ethylene, and 600 sccm of Hydrogen at 900° C. for 10 min, resulting in allocation of oriented. CNTs at specific positions. In another embodiment, the metal electrode definition includes metal electrodes, 10 nm Ti and 30 nm Au, are defined using photolithography and lift off technique. In another embodiment, the spacing between adjacent devices is approximately 20 μm.

As further disclosed herein, the inventors used semiconductive nanotube network as the active channel of biosensors to improve sensitivity. Examples are depicted herein as FIGS. 8, 9 and 10. Devices with high on/off ratio (indication of semiconductivity of the nanotube network) were successfully fabricated using carbon nanotube network where the density of the nanotube was carefully tuned.

The inventors employed semiconductive nanotube network as the active channel of a biosensor, and improved the sensitivity (lowest detection limit) by more than 20 times compared to devices with mixed nanotube (both metallic and semiconductive nanotubes) network. Preparation of such semiconductive network was done by controlling the nanotube density to overcome the percolation limit for semiconductive nanotubes and not to overcome that for metallic nanotubes. This method does not require any pre/post treatments on the devices, and is easily applicable to wafer scale or even larger scale production.

In one embodiment, the present invention provides a biosensor array that utilizes a semiconductive nanotube network as the active channel. In another embodiment, the semiconductive nanotube network provides an improved sensitivity.

In another embodiment, the present invention provides a method of fabricating a nanotube biosensor by growing single-walled carbon nanotubes on a degeneratively doped Si wafer with 500 nm SiO₂ on top via chemical vapor deposition method with Fe nanoparticles formed from ferritin molecules as catalysts. In another embodiment, the nanotube biosensor is made by one or more of the following steps: (1) Diluted solution of ferritin in De-ionized water (D.I. water) is put on the Si/SiO₂ wafer and kept for 1 h at room temperature, resulting in deposition of ferritin molecules onto the substrate; (2) the substrate is washed with D.I. water, followed by calcination in air at 700° C. for 10 min, allowing formation of Fe nanoparticles; (3) after the calcination. the substrate is placed in a quartz tube that was heated to 900° C. in hydrogen atmosphere, and once the temperature reaches 900° C., methane (1300 sccm), ethylene (20 sccm), and hydrogen (600 sccm) are flowed into the quartz tube for 10 min, which yields a CNT network on the substrate; (4) following the growth is patterning of source-drain electrodes, done by photolithography and lift off technique; (5) oxygen plasma is then performed for 1 min in order to etch unwanted CNTs while covering the channel areas with poly(methyl methacrylate) (PMMA). In another embodiment, the metal electrodes are made of 10 nm Cr and 30 nm Au. In another embodiment, the channel width and length of the resultant devices are 5 mm and 100 μm, respectively.

Nanosensor Platforms—Attachment of PDMS Chips to Silicon/Silica Oxide Surfaces

As disclosed herein, the inventors have developed novel techniques to attach PDMS chips to a silicon/silica surface. In the past, attachment of PDMS to silicon/silica surfaces has been achieved mostly via the generation of highly reactive radical species on the PDMS surface via oxygen plasma activation, which can react with OH group present on the silicon/silica surface. In order to react, the OH groups on the silicon/silica must be vertically aligned with the radicals on the PDMS surface. The larger the number of successful reactions, the stronger the adhesion of PDMS to silicon/silica. Thus it may be desirable to have a large surface density of OH groups on Si/SiO₂ and of radicals on the PDMS. The Si/SiO₂ chip at the end of nanowire/nanotube growth process is highly dehydrated with a significantly reduced number of OH groups on its surface. A method to generate a large surface density of OH groups on the dehydrate Si/SiO₂ surface would be highly valuable. As depicted in FIG. 11, the inventors have found that treating the Si/SiO₂ surface with a bifuntional molecule, bearing a silane terminal group (where W, Y. or Z can be a methoxy, ethoxy, methyl, or etc, group) which has binding affinity to Si/SiO₂, at the other terminal another functional group reactive toward activated PDMS (where X can be a NH2, methoxy, etc.), and these two terminals are separated by a carbon chain (with n=0.1, 2, 3, etc.), is useful to covalently bind the surface of silicon/silicon oxide to PDMS chips. Other linker molecules also useful for this purpose would include Si(OH)₄ and Si(OR)₄ as well as alkyl silanes; as used herein, R is an alkyl or H.

In one embodiment, the present invention provides a method of attaching a PDMS microfluidic chip to a silicon and/or silica surface. In another embodiment, the PDMS microfluidic chip is attached by one or more of the following steps: (1) A silane derivative linker molecule is freshly distilled prior to its use; (2) a solution with a concentration of silane derivative in the 2-10% range in methanol is prepared; (3) the silicon/silica surface is cleaned using oxygen plasma (60 Ton O2, 1-10 minute(s)), where if any feature present of the Si/SiO₂ surface is sensitive to 0₂ plasma it can be protected with a layer of PMMA; (4) the silicon/silica surface is submerged in the silane-derivative methanolic solution overnight; (5) unbound silane derivative is washed away with methanol; (6) the PMMA is washed away with hot acetone or anisole; (7) the silicon/silica chip is baked for 1-2 hours at 120° C., and the Si/SiO₂ chip is ready for attachment; (8) the PDMS chip is activated with oxygen plasma (60 Ton O2, 1-10 minute(s)); (9) the activated PDMS chip and derivatized Si/SiO₂ chip are placed in contact with each other and backed at 120° C. for 2 hours; (10) robust attachment of PDMS to silicon/silica is accomplished.

Nanosensor Platforms—Calibration, Tuning and Sensitivity, and Reproducibility

As known in the art, large variation of sensor performance (which determines the reliability of the sensors especially for quantitative analysis) has been a challenge in past efforts to commercialize nanosensors. Although a possible solution to this problem is to make uniform devices, there has been reported difficulty associated with the precise control of the nanomaterial synthesis.

As disclosed herein, the inventors have developed a data analysis method to calibrate the sensor response. Examples are depicted herein as FIGS. 12, 13 and 14. Because the variation of the sensor responses is decreased, and it is a simple analysis method that does not require intensive efforts, the method is complementary to producing uniform devices. The inventors calibrated the response of In₂O₃ nanowire biosensor using parameters extracted from transistor measurements, resulting in decrease in coefficient of variation. The inventors have shown that two parameters can be extracted from transistor measurements that can be used to normalize the responses of In₂O₃ nanowire biosensor. Two definitions of response were employed, one is absolute response, and the other is relative response.

In one embodiment, the present invention provides a method of calibrating a sensor response. In another embodiment, the method of calibrating a sensor response includes extracting parameters from transistor measurements. In another embodiment, the parameters include dIds/dVg and/or dlogIds/dVg. In another embodiment, the sensor is an In₂O₃ nanowire biosensor. In another embodiment, liquid gate measurement is used to calibrate the sensor response. In another embodiment, the calibration includes ΔI and/or ΔG. In another embodiment, the calibration includes ΔI/I and/or ΔG/G.

As further disclosed herein, the inventors tuned the sensitivity of the In₂O₃ nanowire biosensor by using liquid gate. An example is depicted herein as FIG. 15. In spite of the importance of sensitivity as a metric to evaluate the performance of a sensor, there is a need to increase the sensitivity, especially by means of a post-device-fabrication method. In accordance with various embodiments described herein, liquid gate can be used to tune the sensitivity of biosensors by applying different gate voltage to the liquid gate. Similarly, the method is applicable to field effect transistor type biosensors.

In one embodiment, the present invention provides a method of tuning the sensitivity of a biosensor by using a liquid gate. In another embodiment, the biosensor includes an In₂O₃ nanowire. In another embodiment, the liquid gate is used to tune the sensitivity of a biosensor by applying different gate voltage to the liquid gate. In another embodiment, the present invention provides an apparatus for tuning the sensitivity of a biosensor where a chemical cell made of teflon is mounted onto a device and filled with PBS, and a Pt wire is inserted into the PBS and serves as a gate electrode and/or liquid gate.

II. Analysis of Biomarker Signals for the Detection of Disease Analysis of Biomarker Signals for the Detection of Disease—Multimarker Signatures of Disease on Nanosensor Platforms

As disclosed herein, the inventors have created sensor devices, based on nanowires and/or nanotubes, designed for detection and monitoring of a disease and/or condition. The device may recognize the unique molecular signatures arising upon development of a disease, bacterial or viral contamination, allergic reactions, etc. These devices also use the capturing capabilities of any and all capture molecules such as antibodies, PNA, RNA, DNA and protein aptamers, oligonucleotides including RNA and DNA, receptors, ligands, and any other capture molecules that can detect biomolecules. Thus, the detection platform can detect the unique molecular signatures of a specific disease as well as eliminate or reduce the need for multiple tests, thus reducing the total time required to have a final evaluation of the health status of a patient.

In one embodiment, the present invention provides an apparatus for detecting and/or monitoring a disease and/or condition comprising a sensor device bound to a capture molecule, where the capture molecule may recognize a molecular signature associated with a disease and/or condition. In another embodiment, the sensor device includes nanowire and/or nanotube. In another embodiment, the capture molecule and/or molecules may be a polynucleotide, polypeptide, antibody, aptamer, receptor, ligand, or combinations thereof. In another embodiment, the disease and/or condition is cancer.

In one embodiment, the present invention provides a method of detecting and/or monitoring a disease by binding a sensor device to a capture molecule, where the capture molecule may recognize a molecular signature associated with a disease and/or condition. In another embodiment, the sensor device includes nanowire and/or nanotube. In another embodiment, the capture molecule may be a polynucleotide, polypeptide, antibody, aptamer, receptor, ligand, or combinations thereof. In another embodiment, the disease and/or condition is cancer.

In another embodiment, the present invention provides a method of analyzing one or more biomarker signals for the detection of a disease by the following steps, or combinations thereof: (1) Devices based on nanowires or nanotubes are obtained; (2) the surface of the nanomaterial is cleaned; (3) the surface of the each individual device or group of devices on a chip is then functionalized with a particular capture probe; (4) the conductance of the device is monitored over time, while the chip is then placed in contact with the solution under analysis where each single biomarker for a particular disease is captured by its corresponding probe molecule.

Analysis of Biomarker Signals for the Detection of Disease—Peptide Aptamers and Protein Aptamers as Capture Agents for Nanobiosensors

As further disclosed herein, the inventors employed peptide aptamers and/or protein aptamers as capture agents in multiplex detection of the biomarker for a specific disease, as well as microbial/virus detection, in a sensor device based on nanowire/nanotube. Peptide aptamers are sequences of peptides with a defined three dimensional structure that have shown to bind with high affinity and specificity to a particular biological molecule (protein, etc.). A common example consists of a variable peptide attached at both ends to a protein scaffold, and the variable length is typically comprised of 10 to 20 amino acids. The peptide bond (of both peptide and protein aptamers) is stable over a large range of pH values, giving protein aptamers unique robustness. Moreover, peptide and protein aptamers are much smaller in size (2-3 nm and 2-6 KDa) than antibodies causing the captured molecule (analyte) to be spatially closer to the nanowire/nanotube. This analyte-nanowire closeness causes the electric field of the analyte to exert a greater influence on the charge carrier in the device.

In one embodiment, the present invention is a sensor device for multiplex detection of a biomarker for a specific disease, where the capture molecule is a peptide aptamer and/or protein aptamer. In another embodiment, the peptide aptamer and/or protein aptamer is immobilized on the surface of a nanowire.

In another embodiment, the present invention provides a method of fabricating a biosensor device for multiplex detection of biomarkers by the following steps, or combinations thereof: (1) Devices based on nanowires or nanotubes are obtained; (2) The surface of the nanomaterial is cleaned; (3) The surface of the nanomaterial is then functionalized with a linker molecule; (4) The peptide aptamer or protein aptamer is covalently bound to the linker molecule and thus is immobilized on the nanomaterial surface.

Analysis of Biomarker Signals for the Detection of Disease—Methods to Reduce Background Noise

As disclosed herein, background noise can be a significant problem when performing the detection of an analyte in the case in which: (1) The analyte possesses a low net charge. In this case, a technique called sandwich assay can be used to amplify the binding signal. (2) The analyte is present at trace levels in a complex mixture of biomolecules such as blood, serum, or urine. In such mixtures background biomolecules might be present at concentration approaching up to a trillion times the concentration of the analyte. When the analyte is present at trace level, the physiological solution can be preprocessed to remove major serum components (either using pre-processing chromatographic column or by functionalization of the microfluidic channels with “removing agents”) or the surface of the nanomaterial can be functionalized with one or more molecules that diminish any “non-specific” binding event.

In accordance with various embodiments described herein, the physiological solution may be preprocessed by utilizing a solid phase previously modified using capture agents with high affinity for major background components. This solid phase could take on a number of different forms, including beads, modified PDMS microfluidic walls, or a patterned microarray (on a separate chip than the nanosensor array). Importantly, the capture component is mounted to a stationary support, which the sample is passed over before it reaches the nanosensor. Thus, the major background components are removed from the sample, prior to the sample encountering the nanosensor.

In one embodiment, the sandwich assay is used to amplify the signal from a binding event to detect molecules with low net charges. Traditional nanowire biosensing has usually been done with a two-step approach: attachment of probe molecules followed by binding of the target biomolecules. The inventors propose combining a sandwich antibody assay with nanowire biosensing. In another embodiment, the sandwich assay may be performed by one or more of the following steps: First, primary antibodies to target proteins are attached to the NWs. The analyte (containing target proteins) is then delivered to the NWs. After capture of the target proteins, secondary antibodies, directed to the target protein and conjugated to signal enhancer (charged nanoparticles or proteins) are introduced. The secondary antibodies with signal enhancer can increase signal as well as specificity because the target proteins are recognized by two antibodies with the secondary antibodies attached to a signal enhancer. Similarly, in another embodiment, a modified version of the sandwich assay can also be performed to amplify the binding signal when the analyte is a DNA/RNA nucleotide. The hybridized DNA (forming a duplex on the NW surface) can be intercalated using highly charged intercalators such as YOYO1 (as depicted in FIG. 16). Small, highly charged molecules such as YOYO1 can insert in the DNA double helix and their molecular charge influences the device conductance. As readily apparent to one of skill in the art, the sandwich assay would not be restricted to the use of proteins.

In another embodiment, the physiological solution may be preprocessed to remove major biological components; In another embodiment, the physiological solution is preprocessed by utilizing a microfluidic system. In another embodiment, the preprocessing includes a pre-processing chromatographic column for removal of non-specific proteins and molecules. In another embodiment, the physiological solution is pre-processed by attaching antibodies to the major serum proteins to the substrate and/or walls of a PDMS microfluidic channels in order to achieve a “on-chip” removal of non-specific proteins. As disclosed herein, these techniques significantly reduce the concentration of background proteins in the physiological-sample containing the analyte, reducing the risk of false positives and false negatives. As readily apparent to one of skill in the art, the physiological solution is not-restricted in any way to serum. Similarly, the preprocessing of the physiological solution may be utilized in conjunction with a variety of molecules in addition to proteins.

In another embodiment, PEG groups may be added to the nanowire surface to prevent nonspecific bindings of non-specific serum proteins. As disclosed herein, PEG groups on the nanowires will repel proteins that non-specifically might interact with the nanowire surface.

Analysis of Bionmarker Signals for the Detection of Disease—Detection of a DNA/RNA Duplex Using a PNA Capture Probe

As further disclosed herein, capture of double stranded DNA (dDNA) or double stranded RNA (dRNA) may be achieved using either a single strand PNA probe or a double stranded PNA probe immobilized on the surface of a nanowire FET sensor. PNA probes are known to hybridize to double stranded DNA/RNA via “invasion”. An example is depicted herein as FIG. 17. This invasion interaction will be detected by a nanowire/nanotube FET sensor due to the high molecular charge of the captured dDNA/dRNA. PNA probes are known to hybridize to double stranded DNA/RNA via “invasion”. Capturing duplex DNA instead of a single strand DNA allows for a more direct detection by eliminating a pre-analysis step that unfold/melt the dsDNA into two ssDNAs. Another advantage of this strategy is given by the large molecular charge on the duplex DNA whose strong electric field can expert a vast influence on the current carries of the device.

In another embodiment, the invention may detect a polynucleotide by using a PNA capture probe.

As apparent to one of skill in the art, any number of biomarkers may be used in conjunction with various embodiments described herein. Some examples of biomarkers include, but are not limited to, polypeptides, antigens such as glycosylated subunits and lipids, and polynucleotides including microRNA, microsatellite DNA, SNPs, and both genetic and epigenetic. Similarly, as apparent to one of skill in the art, the various embodiments described herein may be used for any number of diseases and conditions. Some examples of diseases and conditions include, but are in no way limited to, cancer, cariac disease, autoimmune disease, endocrine disease, brain disease, reproductive disease, infectious disease including viruses, prions, bacteria, fungi, yeast, and methods to evaluate prenatal status.

Other features and advantages of the invention will become apparent from the following detailed description, which illustrate, by way of example, various features of embodiments of the invention.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1 Nanowire Sensor Fabrication: Interdigitated Electrode

Interdigitated electrodes were used to achieve an easy and reliable fabrication of nanowire field effect transistor biosensors with improved uniformity and yield compared to devices fabricated with non-interdigitated electrodes. The inventors employed interdigitated electrodes to increase the effective channel width for the fabrication of nanobiosensor using bottom up approach. These interdigitated electrodes result in increasing the probability/number of the randomly dispersed nanowires to bridge between source and drain, while keeping same footprint. As a consequence, devices fabricated with interdigitated electrodes have more, uniform performance with higher yield compared to devices with non-interdigitated electrodes, and the yield and uniformity of the devices are comparable to those achieved with assembling assisted techniques. This method has several other advantages such as simplicity, scalability, reproducibility, high throughput, and room temperature processing.

Devices were made using this process on a complete 3 inch wafer, and the resultant yield of good devices exceeds 70%. (the definition of a good device is a device with on/off ratio>10²) The process was repeated several times, and reproducibility of the process was confirmed by the consecutive achievement of comparable yield. The devices showed narrow distribution in threshold voltage (CV 38.5%), confirming the uniformity of the devices.

Example 2 Nanowire Sensor Fabrication: Interdigitated Electrode—Methods of Fabrication

The fabrication consists of three steps: First, In₂O₃ NWs (previously grown on a Si/SiO₂ substrate via a laser ablation process developed previously²) were suspended in isopropanol by sonication. The solution was then dispersed onto a complete 3″ Si/SiO₂ substrate, followed by definition of the Ti/Au source and drain electrodes by photolithography. The interdigitated electrodes were designed to have channel length of 2.5 mm and effective channel width of 500, 780, and 2600 mm.

Example 3 Nanowire Sensor Fabrication: Interdigitated Electrode—Results

The inventors presented an optical micrograph of In₂O₃ NW devices with three different geometries on a complete 3″ wafer, an optical micrograph of the interdigitated electrodes, and a SEM image of an In₂O₃ NW bridging the source and drain, respectively. As disclosed herein, the inventors showed transport characteristics of a good device, where it is shown source-drain current (I_(ds)) versus source-drain voltage (V_(ds)) plots under different gate voltages (V_(g)), and I_(ds) versus V_(g) plots in linear and log scale. The I_(ds) versus V_(ds) plot exhibits transistor behavior similar to MOSFET, and I_(ds) versus V_(g) plot shows good gate dependence of the device with an on/off ratio ˜10⁶. The inventors achieved good device yield >70% by fine-tuning the nanowire density. Also disclosed herein, SEM images are shown of In2O3 nanowire samples with high density and low density. Devices fabricated with these nanowire samples were electrically characterized, and the yield of electrical connection (EC), yield of good devices out of electrically connected source and drain (GD), and total yield of good devices (TGD) were plotted versus the effective channel width. The devices made with high density NWs exhibit 100% EC for the every channel width, while that for devices with low density NWs are from 75% for the smallest channel width to 98% for the longest channel width. This increase in EC for higher density NWs and wider channels can be understood straightforward. On the other hand, the yield of GD (and TGD as a result) decreases as the channel width increases for both high and low density NW samples, and the yields of GD for high density NWs are lower than that for low density NW samples with same channel widths. This may be explained by percolation of “bad” NWs, meaning NWs with little gate dependence, between source and drain electrodes, with analogy to the percolation of metallic nanotube pathway in networked carbon nanotube transistors. NWs with large diameters that were often observed after the growths can be such “bad” NWs, since the effect of gate voltage may not be able to modulate the entire body of the NWs due to the large diameter of the NWs. In addition to the yields, the uniformity of the devices was analyzed. As disclosed herein, the distribution of threshold voltage of the devices made on a Si substrate capped with 50 nm SiO2 with a channel width of 2,600 μm. It exhibits an average value of −0.65 V and a standard deviation of 0.25 V, which yield a coefficient of variance of 38.5%. This is only slightly higher than that of Si NW devices assembled with the langmuir Blodgett method (34.6%).

Example 4 Nanowire Sensor Fabrication: Top-Down Fabrication of Silicon Nanowire Using Single-Crystal Silicon, Poly-Silicon and/or Amorphous Silicon-on-Insulator

The inventors employed poly-silicon and amorphous-silicon as active materials to replace single crystal silicon in Silicon-on-Insulator (SOI) structure to fabricate high-density and uniform arrays of biosensors using a top-down approach. By replacing single crystal silicon in SOI wafer with poly-silicon and amorphous silicon, the cost is reduced significantly, while keeping the advantage of top-down, foundry compatible fabrication that results in high yield and small device-to-device variation. Furthermore, amorphous-silicon can be deposited on unconventional rigid/flexible substrates such as glass and PET, which further reduces the device price.

Example 5 Nanowire Sensor Fabrication: Top-Down Fabrication of Silicon Nanowire Using Single-Crystal/Poly-Crystal/Amorphous Silicon-on-Insulator—Methods of Fabrication

The process consists of the following steps:

-   -   1. Poly-silicon or amorphous-silicon is deposited on appropriate         substrates, or single-crystal silicon-on-insulator wafers are         used as starting substrates.     -   2. Active layer mesa is defined using photolithography and         reactive ion etching (RIE).     -   3. Ion implantation is done to create degenerate lead-in.     -   4. Annealing is done to active the dopants.     -   5. E-beam writing and RIE is used to define nanowires with         desired width.     -   6. Metal contacts are created using photolithography and         lift-off technique.

Example 6 Nanotube Sensor Fabrication: Highly Aligned Carbon Nanotubes

The inventors employed aligned carbon nanotubes to fabricate large, high-density arrays of sensors. The inventors developed a novel and straightforward approach for manufacturable and scalable biosensor arrays based on patterned growth of aligned carbon nanotubes at desired locations. This approach has several advantages over competing techniques in terms of 1) mass production due to the use of conventional fabrication process without e-beam writing, 2) uniform and reproducible device performance due to the use of multiple nanotubes, and 3) deterministic construction of biosensor arrays at specific locations and at any array size. Use of ordered nanotube arrays offer significant advantages, since the orientation control eases and increases the reproducibility of the sensor array fabrication. Furthermore, the architecture is fault tolerant: the destruction of one channel leaves other channels still open, providing a conduction pathway between source and drain. This approach may also be used as the basis for a multiplexed nanobiosensor array fabrication. Biosensor arrays that utilize aligned carbon nanotubes as a semiconductor channel were successfully fabricated and used for the detection of a biomolecules, IgG.

Example 7 Nanotube Sensor Fabrication: Methods of Fabrication

The inventors have fabricated carbon nanotube FET arrays in a multistep process, illustrated herein. The process consists of the following steps:

(1) Catalyst preparation: Quartz substrates were photolithographically patterned to make openings for catalysts. A solution of ferritin (Sigma) in de-ionized (D.I.) water was dropped onto the substrates, and kept for 10 min. The substrates were then rinsed with D.I. water, and the photoresist layer was lifted off in Acetone. The substrate with ferritin particles was calcinated at 700° C. for 10 min to form iron oxide nanoparticles that act as catalysts. (2) Aligned carbon nanotube growth: A chemical vapor deposition (CVD) growth of CNTs was performed with 2,500 sccm of Methane, 10 sccm of Ethylene, and 600 sccm of Hydrogen at 900° C. for 10 min, resulting in allocation of oriented CNTs at specific positions. (3) Metal electrode definition: Finally, metal electrodes (10 nm Ti and 30 nm Au) were defined using photolithography and lift off technique.

Following these procedures, the inventors successfully fabricated aligned nanotube biosensor arrays. The spacing between two adjacent devices was ˜20 μm, and each device was clearly separated as is confirmed from the SEM images showing no nanotubes crossing between two devices.

Example 8 Nanotube Sensor Fabrication: Device Characterization of Aligned Nanotube Devices and IgG Sensing with Anti-IgG Antibody

The inventors have characterized the electrical property of aligned nanotubc devices, and the result is shown herein. The device resistance showed mean value of 154.9 kΩ with a standard deviation of 132.2 kΩ. The distribution can be narrowed down by growing higher density of aligned nanotubes. The inventors have further used those devices to detect IgG antigen. The device was first functionalized with anti-IgG antibody by soaking in a solution of anti-IgG antibody in PBS buffer for 12 h at 4° C. The device was then exposed to a solution of IgG antigen at a concentration of 100 nM, and the device showed a conductance drop of ˜8%. Upon exposure to 7 μM IgG antigen, the device showed further decrease in conductance by ˜7%. This result confirms the successful use of aligned nanotube based biosensor.

Example 9 Nanotube Sensor Fabrication: Use of Semiconductor Nanotube Networks

The inventors used a semiconductive nanotube network as the active channel of biosensors to improve the sensitivity. Single walled carbon nanotube can be categorized into two types, which are metallic and semiconductive nanotubes. It has been shown that semiconductive nanotube is more susceptible to the environment compared to metallic nanotube. This indicates that carbon nanotube based biosensors where the transport is dominated by the one through semiconductive nanotubes has a possibility to exhibit better sensitivity than biosensors where the transport happens through both metallic and semiconductive nanotubes. Based on this idea, the inventors have employed semiconductive nanotube network as the active channel of our biosensor, and improved the sensitivity (lowest detection limit) by more than 20 times compared to devices with mixed nanotube (both metallic and semiconductive nanotubes) network. Preparation of such semiconductive network was done by controlling the nanotube density to overcome the percolation limit for semiconductive nanotubes and not to overcome that for metallic nanotubes. This method does not require any pre/post treatments on the devices, and is easily applicable to wafer scale or even larger scale production. Devices with high on/off ratio (indication of semiconductivity of the nanotube network) were successfully fabricated using carbon nanotube network where the density of the nanotube was carefully tuned.

Example 10 Nanotube Sensor Fabrication: Methods of Fabrication

CNTs were grown on a degenerately doped Si wafer with 500 nm SiO₂ on top via chemical vapor deposition (CVD) method with Fe nanoparticles formed from ferritin molecules as catalysts. The following describes the device fabrication:

(1) Diluted solution of ferritin in De-ionized water (D.I. water) was put on the Si/SiO₂ wafer and kept for 1 h at room temperature, resulting in deposition of ferritin molecules onto the substrate. The substrate was then washed with D.I. water, followed by calcination in air at 700° C. for 10 min, allowing formation of Fe nanoparticles. (2) After the calcination, the substrate placed in a quartz tube was heated to 900° C. in hydrogen atmosphere, and once the temperature reached 900° C., methane (1300 sccm), ethylene (20 sccm), and hydrogen (600 sccm) were flowed into the quartz tube for 10 min, which yields a CNT network on the substrate. (3) Following the growth was patterning of source-drain electrodes, done by photolithography and lift off technique. The metal electrodes were made of 10 nm Cr and 30 nm Au. The channel width and length of the resultant devices were 5 mm and 100 μm, respectively. Oxygen plasma was then performed for 1 min in order to etch unwanted CNTs while covering the channel areas with poly(methyl methacrylate) (PMMA).

It should be noted that the control of CNT density is critical to achieve high on/off ratio devices, which was done by tuning the density of catalyst particles. Typical density of CNTs that yields high on/off ratio devices is shown herein.

Example 11 Nanotube Sensor Fabrication: Device Characteristics

Typical electrical characteristics of such CNT devices are shown herein Source-drain current (Ids) versus source-drain voltage (Vds) under different gate voltage (Vg) are plotted and shows clearly separated curves under different Vg. These clearly separated curves indicate the good sensitivity of the device to the gate voltage, which led to the improved sensitivity of the device as shown later. This good sensitivity to gate voltage can be also observed in the Ids versus Vg curve shown herein. It should be noted that usually CNT devices using a network of mixture of semiconductive and metallic nanotubes show on/off ratio <10.

Example 12 Nanotube Sensor Fabrication: Comparison of Sensitivity of Devices Using Semiconductive Nanotube Network and Mixed Nanotube Network

To verify the advantage of using semiconductive nanotube network over mixed nanotube network, the inventors tested the sensitivity of both devices using Streptavidin (SA) as a model case. The devices were mounted in an electrochemical cell, and the cell was filled with phosphate saline buffer. The conductance of the devices was monitored while being exposed to solutions of SA at different concentrations. The sensing of SA with a device using semiconductive nanotube network is disclosed herein. The device showed ˜1% conductance drop upon exposure to a solution of SA at 100 pM, and further addition of SA at higher concentrations gave large conductance drops. On the other hand, a device using mixed nanotube network showed only negligible response (<<1%) when exposed to 2 nM SA, indicating that the lowest detection limit of mixed nanotube network devices is at least lower by a factor of 20 compared to semiconductive nanotube network devices. Furthermore, the comparison of magnitude of responses to SA at different concentrations revealed the enhanced sensitivity of semiconductive nanotube network devices over mixed nanotube network devices, confirming the advantage of the use of semiconductive nanotube network as biosensors.

Example 13 Attachment of PDMS Chips to Silicon, Silicon Oxide, or Glass Surfaces

The inventors developed a novel technique for attaching PDMS chips to silicone/silica oxide surfaces. In the past, attachment of PDMS to silicon/silica surfaces has been achieved mostly via the generation of highly reactive radical species on the PDMS surface via oxygen plasma activation, which can react with OH group present on the silicon/silica surface. In order to react, the OH groups on the silicon/silica must be vertically aligned with the radicals on the PDMS surface. The larger the number of successful reactions, the stronger the adhesion of PDMS to silicon/silica. Thus is important to have a large surface density of OH groups on Si/SiO2 and of radicals on the PDMS. The Si/SiO2 chip at the end of nanowire/nanotube growth process is highly dehydrated with a significantly reduced number of OH groups on it surface. A method to generate a large surface density of OH groups on the dehydrate Si/SiO2 surface would be highly valuable. As depicted in FIG. 11, the inventors have found that treating the Si/SiO2 surface with a bifuntional molecule, bearing a silane terminal group (where W, Y, or Z can be a methoxy, ethoxy, methyl, or etc. group) which has binding affinity to Si/SiO2, at the other terminal another functional group reactive toward activated PDMS (where X can be a NH2, methoxy, etc.), and these two terminals are separated by a carbon chain (with n=0, 1, 2, 3, etc.), is very useful to covalently bind the surface of silicon/silicon oxide to PDMS chips. Other linker molecules also useful for this purpose would includeSi(OH)₄ and Si(OR)₄ as well as alkyl silanes; as used herein, R is an alkyl or H.

By using any of the aforementioned linker molecules, one can accomplish a very robust attachment of PDMS chip to a silicon/silica surface.

Example 14 Attachment of PDMS Chips to Silicone/Silica Oxide Surfaces: Methods of Fabrication

Methods of fabrication include the following:

-   -   1. The silane derivative linker molecule is freshly distilled         prior to its use.     -   2. A solution with a concentration of silane derivative in the         2-10% range in methanol is prepared.     -   3. The silicon/silica surface is cleaned using oxygen plasma (60         Torr O2, 1-10 minute(s)). If any feature present of the Si/SiO2         surface is sensitive to O2 plasma it can be protected with a         layer of PMMA.     -   4. The silicon/silica surface is submerged in the         silane-derivative methanolic solution overnight.     -   5. Unbound silane derivative is washed away with methanol.     -   6. The PMMA is washed away with hot acetone or anisole     -   7. The silicon/silica chip is baked for 1-2 hours at 120° C. The         Si/SiO2 chip is ready for attachment.     -   8. The PDMS chip is activated with oxygen plasma (60 Torr O2,         1-10 minute(s)).     -   9. The activated PDMS chip and derivatized Si/SiO2 chip are         placed in contact with each other and backed at 120° C. for 2         hours.     -   10. Robust attachment of PDMS to silicon/silica is accomplished.

Example 15 Calibration, Tuning and Sensitivity, and Reproducibility

The inventors calibrated the response of In2O3 nanowire biosensor using parameters extracted from transistor measurements, resulting in decrease in coefficient of variation (CV). The inventors have developed a data analysis method to calibrate the sensor responses. This method has an advantage that it can decrease the variation of the sensor responses. Furthermore, it is a simple analysis method that does not require intensive efforts often needed to make uniform devices. The method is complementary to those to make uniform devices. The inventors have shown that two parameters that can be extracted from transistor measurements can be used to normalize the responses of In2O3 nanowire biosensor. Two definitions of response were employed, one is absolute response, and the other is relative response.

Example 15 Calibration, Tuning and Sensitivity, and Reproducibility: Methods of Fabrication

Extraction of device parameters: To extract parameters used to calibrate the sensor responses, the inventors employed liquid gate measurement. The schematic diagram of the measurement setup is shown herein. A chemical cell made of teflon was mounted onto the device, and filled with phosphate saline buffer (PBS) diluted by 100 times with de-ionized water. A Pt wire was inserted into the buffer, and served as a gate electrode (liquid gate). The voltage to the liquid gate (Vg) was swept from −0.6 V to 0.6 V while applying 0.2 V between source and drain electrodes (Vds), and the conduction through the source and drain (Ids) was monitored as a function of the gate voltage. Atypical Ids-Vg curve is shown herein, plotted in linear scale and in log scale. Using the Ids-Vg curve obtained, the inventors extracted device parameters to normalize the sensor response. They used two parameters, dIds/dVg and dlogIds/dVg to normalize absolute response (defined by Ids before sensing−Ids after sensing at Vg=600 mV) and relative response (defined by Ids before sensing−Ids after sensing divided by Ids before sensing at Vg=600 mV), respectively.

Sensing: The inventors used Streptavidin and Avidin as model cases to verify the validity of their idea. The devices were exposed to a solution of Streptavidin or Avidin (1 μM) in 100 times diluted PBS, and Ids-Vg measurement was done before/after the exposure. Absolute response and relative response were calculated from these curves. A typical Ids-Vg curve before/after the exposure of the device to a solution of Streptavidin, with an inset of a SEM image showing Streptavidin molecules tagged with 10 nm Au nanoparticles captured by an In2O3 nanowire functionalized with biotin, is disclosed herein.

Calibration of sensor response: The inventors successfully normalized the absolute response and relative response by dividing them by dIds/dVg and dlogIds/dVg, respectively. As disclosed herein, the inventors show as an example absolute responses plotted against device identification number together with an average of the response before the calibration. In comparison, the normalized device responses (absolute response/dIds/dVg) showed smaller deviation from the average, which is after performing the calibration. The smaller deviation was verified by taking coefficient of variation (CV), which is one standard deviation divided by the mean. As disclosed herein, CV is shown for the absolute and relative responses before/after the calibration for Streptavidin and Avidin, respectively, where the decreased CV after calibration for every response is shown, in support for the validity of the idea.

Example 16 Calibration, Tuning and Sensitivity, and Reproducibility: Sensitivity Tuning Using Liquid Gate

The inventors tuned the sensitivity of In2O3 nanowire biosensors by using a liquid gate. This method can increase the sensitivity of biosensors, and is applicable to the same type of biosensors, i.e. field effect transistor type biosensors. The inventors have shown that liquid gate can be used to tune the sensitivity of biosensors by applying different gate voltage to the liquid gate.

Liquid gate configuration: The schematic diagram of the measurement setup is shown herein. A chemical cell made of teflon was mounted onto the device, and filled with phosphate saline buffer (PBS) diluted by 100 times with de-ionized water. A Pt wire was inserted into the buffer, and served as a gate electrode (liquid gate).

Sensitivity tuning by liquid gate: The inventors used Streptavidin (SA) as a model cases to test their idea, and checked the sensitivity of one In2O3 nanowire device while keeping the gate voltage (Vg) at different biases. Shown herein is the sensing of SA when Vg=0.4 V. When the device was exposed to SA of 10, 100, and 1,000 nM, the device showed increased normalized conductance by 1, 2, and 10%, respectively. On the other hand, with Vg=0.1 V, the device showed increased normalized conductance by 4, 24, and 47%, respectively, indicating the increased response.

Example 17 Analysis of Biomarker Signals: Multimarker Signatures of Disease on Nanosensor Platforms

The inventors fabricated sensor devices, based on nanowires or nanotubes, designed for the detection and monitor of a specific disease. Each of these devices recognize the unique molecular signatures arising upon development of a disease, bacterial or viral contamination, allergic reactions, etc. These devices may use the capturing capabilities of any and all capture molecules including antibodies, RNA, DNA and protein aptamers, oligonucleotides including RNA and DNA, receptors, ligands, and any other capture molecules that can detect biomolecules. The detection platform can detect the unique molecular signatures of a specific disease. This device eliminates the need for multiple tests thus reducing the total time required to have a final evaluation of the health status of the patient.

Example 18 Analysis of Biomarker Signals: Methods of Use and Fabricating

Methods of use and fabrication include the following:

-   -   1. Devices based on nanowires or nanotubes are obtained. The         number of devices on a single chip is usually a couple of         dozens.     -   2. The surface of the nanomaterial is cleaned by standard         procedure.     -   3. In order to configure the sensor chip to detect a specific         disease, each biomarker for that disease must be detected using         a single sensory chip. Thus, at least one device per chip will         be design to detect a specific biomarker. Techniques for         selective functionalization of nanowire/nanotubes that utilize         either a microfluidic device or electrochemical techniques are         employed for this selective functionalization.     -   4. The surface of the each individual device or group of devices         on a chip is then functionalized with a particular capture         probe.     -   5. The conductance of the device was monitored over time, while         the chip is then placed in contact with the solution under         analysis where each single biomarker for a particular disease is         captured by its corresponding probe molecule.

Example 19 Analysis of Biomarker Signals: Peptide and/or Protein Aptamer as Capture Agents or Nanosensors

The inventors employed peptide aptamers and/or protein aptamers as capture agents in multiplex detection of the biomarker for a specific disease, as well as microbial/virus detection, in a sensor device based on nanowire/nanotube. Peptide aptamers are sequences of peptides with a defined three dimensional structure that have shown to bind with high affinity and specificity to a particular biological molecule (protein, etc.). An example may consist of a variable peptide attached at both ends to a protein scaffold, and the variable length is typically comprised of 10 to 20 amino acids. The peptide bond (of both peptide and protein aptamers) is stable over a large range of pH values, giving protein aptamers unique robustness. Moreover, peptide and protein aptamers are much smaller in size (2-3 nm and 2-6 KDa) than antibodies causing the captured molecule (analyte) to be spatially closer to the nanowire/nanotube. This analyte-nanowire closeness causes the electric field of the analyte to exert a greater influence on the charge carrier in the device. The inventors have demonstrated that protein aptamers do work as capture molecules when immobilized on the surface of nanowire in a biosensor.

Example 20 Analysis of Biomarker Signals: Methods of Fabricating Peptide and/or Protein Aptamer as Capture Agents or Nanosensors

Peptide and/or Protein aptamers may be fabricated as capture agents or nanosensors by the following:

-   -   1. Devices based on nanowires or nanotubes are obtained     -   2. The surface of the nanomaterial is cleaned by standard         procedure     -   3. The surface of the nanomaterial is then functionalized with a         linker molecule     -   4. The peptide aptamer or protein aptamer is covalently bound to         the linker molecule thus is immobilized on the nanomaterial         surface.     -   5. Once the device is configured with the appropriate capture         molecule, the device is ready to be used as a sensor.

Example 21 Analysis of Biomarker Signals: Methods to Reduce Background Noise—Sandwich Assay

(1) Devices based on nanowires or nanotubes are obtained. The number of devices on a single chip is usually a couple of dozens. (2) The surface of the nanomaterial is cleaned by standard procedure. (3) In order to configure the sensor chip to detect a specific disease, each biomarker for that disease must be detected using the sensory chip. Thus, at least one device per chip will be design to detect a specific biomarker. Techniques for selective functionalization of nanowire/nanotubes that utilize either a microfluidic device or electrochemical techniques are employed for this selective functionalization. (4) The surface of the each individual device or group of devices on a chip is then functionalized with a particular capture probe. (5) The conductance of the device was monitored over time, while the chip is then placed in contact with the solution under analysis where each single biomarker for a particular disease is captured by its corresponding probe molecule. (6) The analyte solution is washed away and replaced with PBS buffer. (7) A secondary ligand that also bind to the captured analyte is flown over the sensor surface. This secondary ligand can be an aptamer or an antibody and it labeled with a highly charged tag or magnetic nanoparticle. The purpose of this tag is to increase the signal due to the binding of the analyte to the nanowire. (8) A step to cross link the analyte to the capture probe can be added to the procedure so the analyte is also covalently bound to the nanowire/nanotube device. This cross linking step utilizes small, highly reactive molecules such as glutaraldehyde or formaldehyde. (9) The DNA analyte is allowed to hybridize with the DNA/PNA probe on the nanowire surface. (10) The solution containing the analyte is washed away with PBS buffer until a stable baseline is obtained. (11) A solution containing the intercalating molecule in PBS is flown over the sensor. The intercalator would insert in the PNA-PNA or DNA-DNA double helix. Depending on the type of charge carried by the intercalator, the conductance of the nanowire may increase or decrease (the intercalator may case either a carrier accumulation or depletion).

Example 22 Analysis of Biomarker Signals: Reduction of Background Noise—Remove Major Biological Components

(1) Build into the microfluidic system a pre-processing chromatographic column for removal of non-specific proteins. (2) Attach antibodies to the major serum proteins to the walls of the PDMS microfluidic channels in order to achieve “on-chip” removal of non-specific proteins: (a) The PDMS microfluidic chip is cleaned by oxygen plasma; (b) The PDMS chip is aligned and gently pressed against the silicon chip surface; (c) The PDMS + sensor chip is baked at 80° C. for at least 2 hours; (d) Covalently attach captured molecules to PMDS (See, for example, Curreli, et al., “Real time, label free detection of biological entities using nanowire based field effect transistors;” IEEE 2008).

Alternatively: (1) the solid phase is functionalized with capture agents (selectively binding to the background molecules to be removed from the solution). This solid phase can be either on a separate chip or on the sidewalls of the microfluidic device. (2) The physiological solution is flown over the modified solid phase prior to get in contact with the nanosensors.

Example 23 Analysis of Biomarker Signals: Reduction of Background Noise—Addition of PEG Groups to Nanowire/Nanotube Surfaces

(1) The surface of the nanomaterial is cleaned by standard procedure. (2) The surface of nanowire/nanotubes is coated with a linker molecule. (3) The linker molecule may be further activated for bioconjugation. (3) The surface of the each individual device or group of devices on a chip is then functionalized with a particular capture probe. (4) The capture probe, being a biomolecule of relatively large size when compared to PEG short sequences, won't react with all the possible activated linker molecules on the surface. (5) A PEG chain with a terminal reactive group can be attached to the nanowires/nanotubes using the unreactive surface site, resulting in a passivation of the nanomaterial surface with PEG as well as capture molecules.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below. 

1. A nanosensor comprising: a nanomaterial configured for electrical signaling; and one or more capture agents distributed on a surface of the nanomaterial, wherein the nanosensor is configured such that binding of a target molecule to one of the one or more capture agents causes a change in electrical signaling.
 2. The nanosensor of claim 1, wherein the change in electrical signaling is a change in conductance, current, transconductance, capacitance, threshold voltage, or combinations thereof.
 3. The nanosensor of claim 1, wherein the capture agent comprises a polynucleotide and/or polypeptide.
 4. The nanosensor of claim 1, wherein the capture agent comprises an aptamer, a receptor, a ligand, or a combination thereof.
 5. The nanosensor of claim 1, wherein the nanomaterial comprises a carbon nanotube.
 6. The nanosensor of claim 1, wherein the nanomaterial is fabricated by patterned growth of carbon nanotubes.
 7. The nanosensor of claim 1, wherein the nanomaterial comprises an In₂O₃ nanowire.
 8. The nanosensor of claim 1, wherein the change in transconductance is calibrated by liquid gate measurement.
 9. The nanosensor of claim 1, wherein the target molecule comprises an analyte.
 10. The nanosensor of claim 1, wherein the target molecule comprises a biomolecule.
 11. The nanosensor of claim 10, wherein the presence or absence of the biomolecule is indicative of a molecular signature associated with a disease.
 12. A complementary detection system, comprising: an orthogonal functionalization of a nanomaterial with a substrate.
 13. The complementary detection system of claim 12, wherein the nanomaterial comprises a carbon nanotube and/or an In₂O₃ nanowire.
 14. The complementary detection system of claim 12, wherein the substrate comprises Si/SiO₂.
 15. A method of preparing a biosensor to detect the presence of a molecular signature associated with a disease, comprising: providing a biosensor comprising one or more pairs of interdigitated source and drain electrodes; and fabricating a plurality of nanowires on the one or more pairs of interdigitated source and drain electrodes.
 16. The method of claim 15, wherein the nanowire comprises In₂O₃.
 17. The method of claim 16, wherein the In₂O₃ was grown on a substrate.
 18. The method of claim 15, wherein the one or more interdigitated source and drain electrodes each have a channel length of between 1 micron and 100 microns and a channel width of between 100 microns and 1000 microns.
 19. The method of claim 15, wherein the interdigitated source and drain electrodes each have a channel length of about 2.5 microns and a channel width of about 500, about 780, and/or about 2600 microns.
 20. A method of preparing a biosensor array, comprising: placing a quantity of poly-silicon and/or a quantity of amorphous-silicon on an insulating substrate; and incorporating the quantity of poly-silicon and/or the quantity of amorphous-silicon as a component of the biosensor array.
 21. A biosensor array, comprising: a thin-film semiconductor patterned into one or more nanowires.
 22. A nanosensor platform, comprising: a field effect, transistor configured with a plurality of interdigitated electrodes and nanowire, and a poly/amorphous silicon-on-insulator, wherein the poly/amorphous silicon-on-insulator is a component of the field effect transistor.
 23. The nanosensor platform of claim 22, wherein the nanowire comprises In₂O₃.
 24. A method of fabricating a nanotube biosensor, comprising: preparing a catalyst; growing aligned nanotubes by utilizing prepared catalyst; and defining metal electrodes separated by aligned nanotubes.
 25. The method of claim 24, wherein growing aligned nanotubes comprises a chemical vapor deposition growth of the nanotube with methane, ethylene, hydrogen and/or CO as feedstock.
 26. The method of claim 24, wherein growing aligned nanotubes comprises using sapphire and/or quartz as a substrate.
 27. A method of attaching elastomer polydimethylsiloxane to a silicon/silica surface, comprising: treating the silicon/silica surface with a linker molecule; and attaching the elastomer polydimethylsiloxane to the silicon/silica surface.
 28. The method of claim 27, wherein the linker molecule comprises silicic acid and/or alkyl silane.
 29. The method of claim 27, wherein the linker molecule comprises a silicon compound.
 30. A method of calibrating the response of a nanosensor platform, comprising: extracting one or more electronic properties of the nanosensor platform; and calibrating the response from the one or more electronic properties extracted from the nanosensor platform.
 31. The method of claim 30, wherein one of the one or more electronic properties comprises transconductance.
 32. The method of claim 30, wherein transconductance is defined by dividing by dIds/dVg.
 33. The method of claim 30, wherein transconductance is defined by dividing by dlogIds/dVg.
 34. An apparatus for detecting and/or monitoring a disease, comprising: a nanomaterial; and a plurality of capture molecules bound to the nanomaterial, wherein the plurality of capture molecules are configured for recognizing one or more biomolecules associated with a molecular signature of the disease.
 35. The apparatus of claim 34, wherein the capture molecule comprises a polypeptide.
 36. The apparatus of claim 34, wherein the capture molecule comprises a polynucleotide.
 37. The apparatus of claim 34, wherein the capture molecule comprises an aptamer.
 38. The apparatus of claim 34, wherein the capture molecule comprises a polynucleotide complex.
 39. A method of determining the presence of a disease in an individual from whom a sample is obtained, comprising: removing background noise from the sample; providing a nanosensor device configured to detect the presence or absence of a multimarker signature of the disease; and contacting the nanosensor device with the sample to determine the presence or absence of the multimarker signature of the disease, wherein the presence of the multimarker signature is indicative of the disease.
 40. The method of claim 39, comprising removing the background noise by functionalization of the nanosensor device with one or more molecules that prevent binding of a nontarget entity.
 41. The method of claim 39, comprising removing the background noise by amplifying binding signals of the nanosensor device.
 42. The method of claim 39, comprising amplifying binding signals of the nanosensor device using a sandwich assay.
 43. The method of claim 39, comprising removing the background noise by preprocessing the sample to remove a major interfering component.
 44. A method of treating a disease, comprising: providing a nanosensor device configured to detect the presence or absence of a molecular signature of the disease; contacting the nanosensor device with the sample to determine the presence or absence of the molecular signature of the disease; and treating the disease.
 45. A method of improving the sensitivity of a nanosensor, comprising: providing a nanosensor; and performing biosensing measurements by liquid gate voltage and/or back gate voltage to improve the sensitivity of the nanosensor.
 46. A method of preparing a biosensor array, comprising: placing a quantity of semiconductor film on a substrate; and incorporating the quantity of semiconductor film as a component of the biosensor array.
 47. The method of claim 46, wherein the semiconductor film comprises single-crystal silicon.
 48. The method of claim 46, wherein the semiconductor film comprises poly-crystal and/or amorphous silicon. 